Endocrine effects of cholera enterotoxin (CET) on male gonads were investigated in normal and hypophysectomized rats. After intratesticular injection of 5μg of CET in the bilateral testes of normal rats, serum testosterone concentration remarkably increased after 24 hr, remained significantly elevated for at least3 days and returned to the control level in7days. Serum LH level decreased in the undetectable range after1-3days; serum FSH level also significantly decreased after3days. Both gonadotropin levels increased28days after the injection, when the CET-injected testis decreased in weight and was accompanied by marked loss of germinal cells.
When 5μg of CET was injected intratesticularly in the bilateral testes of hypophysectomized rats, adenylate cyclase activity of a CET-injected testis was remarkably stimulated after6hr, remained four times elevated for at least3days and returned to the control level in7days. In relatively good accordance with the increase in adenylate cyclase activity, testosterone content remarkably enhanced in the CET-injected testis.
These in vivo data indicate that the intratesticular injection of CET prolongedly stimulates the adenylate cyclase activity of testicular cells including Leydig cells and increases testosterone production, and suggest that the prolonged enzyme stimulation results in the sustained elevation of serum testosterone concentration for at least 3 days, causing the stimulation of the negative feedback mechanism ofhypophysealtesticular axis to decrease serum LH levels in the undetectable range.

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Recent reports on the binding of glucocorticoid-receptor complexes to rat liver nuclei suggested the presence of components which inhibited the binding. The inhibitory component (s) of the receptor translocation was observed not only in the cytosol of the liver but also in cytosols of the kidney, the spleen and the thymus. The cytoplasmic levels of the inhibitor in these tissues were not modified by the administration of Dexamethasone (DEX). The liver inhibitor was macromolecular and clearly separated from the DEX-receptor complex on DEAE-cellulose chromatography. The mechanism of the inhibition seemed to be an interactionbetween the inhibitor and the steroid-receptor complex. In addition, the inhibition seemed to be less specific for the bindings of different steroid-receptor complexes to nuclei. The bindings of hepatic ³H-DEX-receptor complex by nuclei derived from livers ofadrenalectomized and DEX-treated rats, in the presence or absence of the translocation inhibitor, were similar.

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Simultaneous determination of unconjugated16α-thydroxypregnenolone (16αOHPreg), 16α-hydroxyprogesterone (16αOH-Prog) and16α-hydroxydehydroepiandrosterone (16αOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three16α-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16αOH-Preg in the umbilical arterial plasma increased from11.2±3.1 at24weeks to29.7±12.0ng/ml at term, 16αOH-Prog from15.5±3.2to34.3±11.0 ng/ml and16±OH-DHEA from5.1±1.2to5.9±1.0ng/ml. In the anencephalic neonates, only 16αOH-Preg showed an increase pattern under ACTH priming. 16α-OH-Preg levels for normal full term neonates remain relatively constant at the first 24hr and show a slight decrease at3days post partum. In small full term neonates, 16αOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first5days post partum. 16αOH-Prog and 16αOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first24hr. Comparison with findings of the three16α-hydroxysteroids in fetal and neonatal plasma is discussed.

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Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of10⁹M^-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the ³H-dihydrotestosterone-IgG fraction complex, while binding of ³H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.

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Five-day-old female rats were androgenized with a single dose of 100μg (TP100A) or 1000μg (TP1000A) testosterone propionate. The androgenized adult rats together with normal rats were tested for prolactin and TSH responses to synthetic TRH. Five μg/kg of TRH was injected iv4times at2hr intervals, and serum samples were obtained5min after each injection. The prolactin release by exogenous TRH in normal rats was less marked. In TP1000A rats, the basal prolactin level in the serum was higher than that in normal or TP100A rats and a further elevation was induced by the1st injection of TRH. In TP1, 00A rats, the serum prolactin level increased five-fold more than the maximal level in normal rats by the1st and 2nd injections, while the pituitary prolactin content showed a less marked decline and a tendency to increase after the4th injection, suggesting an enhanced release and production of prolactin by TRH in TP100A rats. TRH injection stimulated TSH release in androgenized rats as well as in normal rats. The magnitude in the TSH release was TP1000A>normal≥TP100A. The TSH contents in the pituitary after TRH injection tended to decrease in TP1000A and normal rats but not in TP100A rats. In TP100A rats, increases of TSH content were evident by the3rd and4th TRH injections. The weight of the anterior pituitary decreased in normal and TP1000A rats but tended to increase in TP100A rats after the treatment with TRH. The present results indicate that not only lactotrophs but also thyrotrophs in the pituitary became very sensitive to exogenous TRH by neonatal androgenization. A marked difference in the responsiveness to exogenous TRH between TP100A and TP1000A rats could be due to the fact that the alterations in the hypothalamus and secondarily in the ovary were induced by testosterone propionate in the dose-dependent manner.

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We have studied the effect of two inhibitors of prostaglandin synthesis on the basal and TRH-stimulated plasma TSH levels in the rat. Animals were injected sc daily with indomethacin (3mg/0.5ml) or aspirin (16-30mg/0.5ml) for3days. The plasma T₄ and T₃ were consistently lower in the indomethacin or aspirin groups than in the controls, while the basal TSH levels did not change. Indomethacin treatment significantly potentiated the TSH response to synthetic TRH (200ng, iv) in intact and thyroidectomized rats. The pituitary TSH content was markedly increased by indomethacin, while hypothalamic TRH content did not change. In contrast, aspirin inhibited the TSH response to TRH in intact rats, when pituitary TSH content decreased significantly. No potentiation by aspirin of TRH-stimulated TSH response in thyroidectomized rats was observed.
The increased sensitivity of plasma TSH response to exogenous TRH in the indomethacin group is presumably due to higher pituitary TSH content than in the controls. The action of indomethacin appears to be mediated, at least in part, at the pituitary level. In addition, there is a dissociation between the action of indomethacin and the action of aspirin in the TSH response to TRH.

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Oral glucose tolerance tests (OGTT) were performed on108patients with hyperthyroidism who were suspected, from their clinical records, of having abnormalities in carbohydrate metabolism. Of these, intravenous glucose tolerance tests (IVGTT) were performed on24patients and intravenous tolbutamide tests (IVTT) on34. Five control subjects were included in the study. In OGTT, an oxyhyperglycemic pattern (OH) was observed in31patients, a borderline pattern in13, and a diabetic pattern with FBS<120mg/dl (Da) in31, FBS: 120-139mg/dl (Db) in11, and FBS≥140mg/dl (Dc) in 22.
Insulin secretory response (ISR) during OGTT increased in the OH group, slightly increased in the Da group, and decreased in the Db and Dc groups, compared with the control group. The insulinogenic index (ΔIRI/ΔBS·E30') during OGTT was higher in the OH group but was significantly lower in the Da, Db, Dc groups than in the control group.
K-value and insulinogenic index (ΔIRI/ΔBS·E3') during IVGTT were2.3±0.45 and 0.10±0.07 (mean±SD, n=5) in the control group, 2.81±1.66 and 0.56±0.25 (n=6) in the OH group, 1.51±0.29 and 0.25±0.15 (n=9) in the Da group, 0.88±0.32 and 0.04±0.03 (n=5) in the Db group, and 0.75±0.11 and 0.00±0.11 (n=4) in the Dc group. While ISR significantly increased in the OH and Da groups during IVTT, the fall of the blood glucose level after tolbutamide administration was similar or suppressed, compared with the control group. Although ISR decreased after the patients had been made euthyroid, there was an increase in glucose disappearance in almost all of the patients.
These findings suggest the following:(1) patients who have an ISR capacity in accordance with that required in a hyperthyroid state may be included in the OH group and patients who do not have it, although they have a sufficient insulin secretory capacity in an euthyroid state, may be included in the Da group;(2) ISR may decrease in most hyperthyroid patients with FBS≥120mg/dl;(3) there may be peripheral insulin resistance in hyperthyroidism.

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The effect of prostaglandin E₁ (PGE₁) on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was studied in the hypertensive subjects treated with without75mg indomethacin or60mg propranolol for a week. Subsequent to the treatment with indomethacin for a week, PRA and PAC levels were decreased as compared to the control, without changes in the blood pressure and heart rate. During the infusion of PGE₁, the blood pressure was decreased and the pulse rate was increased. PRA and PAC levels were also elevated. These changes of parameters were not different between the control and the indomethacin-treated subjects. PRA and PAC were suppressed after the treatment with propranolol. With the infusion of PGE₁, the level of PRA was not significantly elevated, while, PAC was significantly increased by the infusion of100ng/Kg/min of PGE₁. During the infusion of PGE₁, the blood pressure was decreased while the pulse rate was increased in the subjects treated with propranolol. However, the elevation of the pulse rate was less remarkable than the control. These data indicate that PGE, have important roles in the regulation of the release of renin and aldosterone. These findings also suggest that PGE₁may act to stimulate the secretion of aldosterone in man.

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Bilateral ligation of both the submandibular and parotid ducts of adult normal and mutant hyperinsulinemic diabetic mice resulted in a significant hypoglycemic effect. Therefore, we postulated that duct ligation may result in the removal of hyperglycemic factor (Hoshino et al., 1976) rather than a change in insulin sensitivity. Indeed, no change in specific binding of, 25I-insulin was observed in membrane fractions from several tissues obtained from mice of either sex or strains before and after duct ligation. After slices of the submandibular gland were incubated for 4hr in Eagle's medium, an aliquot of the culture medium was injected i. p.into normal adult mice. A significant hyperglycemic effect was observed in30min in the injected animals. Eluates obtained by gel filtration of the crude extract of the submandibular gland were injected into normal adult mice, and hyperglycemia ensued. Thus, it is postulated that ligation of salivary ducts results in glandular atrophy and disappearance of the hyperglycemic factor which in turn leads to hypoglycemia and amelioration of diabetes mellitus, particularly of hyperinsulinemic type.

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Simple method for the collection of large numbers of viable pancreatic islets from normal rats is described. This method involves collagenase digestion of the pancreas, followed by the use of Ficoll-Conray discontinuous gradient for the separation of isolated islets from unwanted acinal debris.
Ficoll-Conray A solution was prepared by mixing undialyzed 12.5% Ficoll and 33.4% Conray in the ratio 2: 1, to make a specific gravity of 1.095 and osmolarity of 396 mOsm/l. The solution B, C, and D, with specific gravities 1.084, 1.072, and 1.048, respectively were obtained by diluting A solution with distilled water.
Using Ficoll-Conray gradient, about200viable islets which maintained excellently their morphology and function, were collected consistently from a young adult rat pancreas.

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The existence of specific progesterone-binding sites with a high affinity and a limited capacity was demonstrated by [³H] progesterone exchange in the nuclear fractions of the pituitary and hypothalamus as well as in those of the oviduct magnum in the hen.

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Thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) concentrations in human milk were measured by radioimmunoassay in 114 samples obtained. from 1 week to 8 months postpartum. Several assay systems applied for the determination of serum thyroid hormone concentration were proved to be unsuitable for human milk, and the method of separating free and antibody-bound hormone by polyethylene glycol was also inappropriate for milk specimens, which tended to give a falsely high value. The binding affinity of T4to milk was lower than that to serum protein, on which 8-anilino-1-naphthalene sulfonic acid showed no remarkable effect. In spite of the high sensitivity of 100pg/tube in T4 assay system, no immunoassayable T4 was detected in all samples with or without ethanol extraction and trypsin hydrolysates of milk. In contrast, T3 was present in a measurable amount in most of the samples, the mean±SD value of which was 10±9ng/100ml, and those in colostrum were significantly higher than those in matured milk (P<0.01), whereas rT3was not detectable in 76 samples tested.
These results indicate that permeability of thyroid hormones through the mammary gland is different between T4 and T3 as well as in placental transport, and human milk can not be a source of thyroxine supply for the breast-fed infant.

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A highly specific tritium labeled oxytocin (³H-OT) was synthesized utilizing the method of catalytic substitution of halogen for hydrogen. The specific activity of 3H-OT was19Ci/mM and the biologic activity was 350U/mg, which was sufficient for the OT radioreceptor assay. The maximum % uptake of ³H-OT in the human myometrium was observed in 20, 000×g pellets under the optimal conditions of pH7.4, at20°C and the incubation time of 90min and it was augmented in the presence of Mn⁺⁺. It was also observed from the Scatchard plot, that the binding site of OT in the human myometrial specimens was a single type within the range of OT concentration of 0.4nM to 1.6nM. The dissociation constants (Kd) and the number of binding sites (NBS) showed a relative increase as gestation advance. The apparent Kd of term pregnancies was 1.25×10^-9M regardless of the presence or absence of labor pains, while the NBS of term pregnancies with and without labor pain was 1.2×10^-12 and 4.7×10^-12moles/mg protein, respectively.

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¹²⁵I-Labeled hGH was bound to liver plasma membranes which were obtained from female rats. The binding was displaced by hGH, hPRL, bPRL, rPRL and bGH but not by rGH. This result indicated that hGH was bound to lactogenic binding sites in rat livers. After hypophysectomy, the binding was markedly decreased. Treatment of hypophysectomized rats with hGH (80μg/day) for10days increased the binding sites for hGH. These binding sites were different from those found in normal female rat livers because of their high affinity and specificity for hGH. These results indicate that hGH induces specific binding sites for hGH in rat livers.

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